Centrobin-mediated Regulation of the Centrosomal Protein 4.1-associated Protein (CPAP) Level Limits Centriole Length during Elongation Stage

Microtubule-based centrioles in the centrosome mediate accurate bipolar cell division, spindle orientation, and primary cilia formation. Cellular checkpoints ensure that the centrioles duplicate only once in every cell cycle and achieve precise dimensions, dysregulation of which results in genetic instability and neuro- and ciliopathies. The normal cellular level of centrosomal protein 4.1-associated protein (CPAP), achieved by its degradation at mitosis, is considered as one of the major mechanisms that limits centriole growth at a predetermined length. Here we show that CPAP levels and centriole elongation are regulated by centrobin. Exogenous expression of centrobin causes abnormal elongation of centrioles due to massive accumulation of CPAP in the cell. Conversely, CPAP was undetectable in centrobin-depleted cells, suggesting that it undergoes degradation in the absence of centrobin. Only the reintroduction of full-length centrobin, but not its mutant form that lacks the CPAP binding site, could restore cellular CPAP levels in centrobin-depleted cells, indicating that persistence of CPAP requires its interaction with centrobin. Interestingly, inhibition of the proteasome in centrobin-depleted cells restored the cellular and centriolar CPAP expression, suggesting its ubiquitination and proteasome-mediated degradation when centrobin is absent. Intriguingly, however, centrobin-overexpressing cells also showed proteasome-independent accumulation of ubiquitinated CPAP and abnormal, ubiquitin-positive, elongated centrioles. Overall, our results show that centrobin interacts with ubiquitinated CPAP and prevents its degradation for normal centriole elongation function. Therefore, it appears that loss of centrobin expression destabilizes CPAP and triggers its degradation to restrict the centriole length during biogenesis.     

Molecular Architecture of the Centriole Proteome: The Conserved WD40 Domain Protein POC1 Is Required for Centriole Duplication and Length Control

Centrioles are intriguing cylindrical organelles composed of triplet microtubules. Proteomic data suggest that a large number of proteins besides tubulin are necessary for the formation and maintenance of a centriole''s complex structure. Expansion of the preexisting centriole proteome from the green alga Chlamydomonas reinhardtii revealed additional human disease genes, emphasizing the significance of centrioles in normal human tissue homeostasis. We found that two classes of ciliary disease genes were highly represented among the basal body proteome: cystic kidney disease (especially nephronophthisis) syndromes, including Meckel/Joubert-like and oral-facial-digital syndrome, caused by mutations in CEP290, MKS1, OFD1, and AHI1/Jouberin proteins and cone-rod dystrophy syndrome genes, including UNC-119/HRG4, NPHP4, and RPGR1. We further characterized proteome of the centriole (POC) 1, a highly abundant WD40 domain-containing centriole protein. We found that POC1 is recruited to nascent procentrioles and localizes in a highly asymmetrical pattern in mature centrioles corresponding to sites of basal-body fiber attachment. Knockdown of POC1 in human cells caused a reduction in centriole duplication, whereas overexpression caused the appearance of elongated centriole-like structures. Together, these data suggest that POC1 is involved in early steps of centriole duplication as well as in the later steps of centriole length control.     

Centrobin-Centrosomal Protein 4.1-associated Protein (CPAP) Interaction Promotes CPAP Localization to the Centrioles during Centriole Duplication

Centriole duplication is the process by which two new daughter centrioles are generated from the proximal end of preexisting mother centrioles. Accurate centriole duplication is important for many cellular and physiological events, including cell division and ciliogenesis. Centrosomal protein 4.1-associated protein (CPAP), centrosomal protein of 152 kDa (CEP152), and centrobin are known to be essential for centriole duplication. However, the precise mechanism by which they contribute to centriole duplication is not known. In this study, we show that centrobin interacts with CEP152 and CPAP, and the centrobin-CPAP interaction is critical for centriole duplication. Although depletion of centrobin from cells did not have an effect on the centriolar levels of CEP152, it caused the disappearance of CPAP from both the preexisting and newly formed centrioles. Moreover, exogenous expression of the CPAP-binding fragment of centrobin also caused the disappearance of CPAP from both the preexisting and newly synthesized centrioles, possibly in a dominant negative manner, thereby inhibiting centriole duplication and the PLK4 overexpression-mediated centrosome amplification. Interestingly, exogenous overexpression of CPAP in the centrobin-depleted cells did not restore CPAP localization to the centrioles. However, restoration of centrobin expression in the centrobin-depleted cells led to the reappearance of centriolar CPAP. Hence, we conclude that centrobin-CPAP interaction is critical for the recruitment of CPAP to procentrioles to promote the elongation of daughter centrioles and for the persistence of CPAP on preexisting mother centrioles. Our study indicates that regulation of CPAP levels on the centrioles by centrobin is critical for preserving the normal size, shape, and number of centrioles in the cell.     

Structural Analysis of the G-Box Domain of the Microcephaly Protein CPAP Suggests a Role in Centriole Architecture

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LUBAC regulates ciliogenesis by promoting CP110 removal from the mother centriole

Primary cilia transduce diverse signals in embryonic development and adult tissues. Defective ciliogenesis results in a series of human disorders collectively known as ciliopathies. The CP110–CEP97 complex removal from the mother centriole is an early critical step for ciliogenesis, but the underlying mechanism for this step remains largely obscure. Here, we reveal that the linear ubiquitin chain assembly complex (LUBAC) plays an essential role in ciliogenesis by targeting the CP110–CEP97 complex. LUBAC specifically generates linear ubiquitin chains on CP110, which is required for CP110 removal from the mother centriole in ciliogenesis. We further identify that a pre-mRNA splicing factor, PRPF8, at the distal end of the mother centriole acts as the receptor of the linear ubiquitin chains to facilitate CP110 removal at the initial stage of ciliogenesis. Thus, our study reveals a direct mechanism of regulating CP110 removal in ciliogenesis and implicates the E3 ligase LUBAC as a potential therapy target of cilia-associated diseases, including ciliopathies and cancers.     

Cep97 and CP110 suppress a cilia assembly program

Mammalian centrioles play a dynamic role in centrosome function, but they also have the capacity to nucleate the assembly of cilia. Although controls must exist to specify these different fates, the key regulators remain largely undefined. We have purified complexes associated with CP110, a protein that plays an essential role in centrosome duplication and cytokinesis, and have identified a previously uncharacterized protein, Cep97, that recruits CP110 to centrosomes. Depletion of Cep97 or expression of dominant-negative mutants results in CP110 disappearance from centrosomes, spindle defects, and polyploidy. Remarkably, loss of Cep97 or CP110 promotes primary cilia formation in growing cells, and enforced expression of CP110 in quiescent cells suppresses their ability to assemble cilia, suggesting that Cep97 and CP110 collaborate to inhibit a ciliogenesis program. Identification of Cep97 and other genes involved in regulation of cilia assembly may accelerate our understanding of human ciliary diseases, including renal disease and retinal degeneration.     

Double life of centrioles: CP110 in the spotlight

Centrioles lead an important double life: they can give rise to the centrosome or convert to basal bodies and template cilia. Little is known about the control of centriole fate. Spektor and colleagues have now identified a centriolar complex, composed of CP110 and CEP97, which inhibits centriole to basal body conversion, preventing cilia formation. This work paves the way to understanding centriole and cilia biogenesis, which are two processes misregulated in human diseases, such as cancer and polycystic kidney disease.     

Centrin2 regulates CP110 removal in primary cilium formation

Primary cilia are antenna-like sensory microtubule structures that extend from basal bodies, plasma membrane–docked mother centrioles. Cellular quiescence potentiates ciliogenesis, but the regulation of basal body formation is not fully understood. We used reverse genetics to test the role of the small calcium-binding protein, centrin2, in ciliogenesis. Primary cilia arise in most cell types but have not been described in lymphocytes. We show here that serum starvation of transformed, cultured B and T cells caused primary ciliogenesis. Efficient ciliogenesis in chicken DT40 B lymphocytes required centrin2. We disrupted CETN2 in human retinal pigmented epithelial cells, and despite having intact centrioles, they were unable to make cilia upon serum starvation, showing abnormal localization of distal appendage proteins and failing to remove the ciliation inhibitor CP110. Knockdown of CP110 rescued ciliation in CETN2-deficient cells. Thus, centrin2 regulates primary ciliogenesis through controlling CP110 levels.     

Daughter Centriole Elongation Is Controlled by Proteolysis

The centrosome is the major microtubule-organizing center of most mammalian cells and consists of a pair of centrioles embedded in pericentriolar material. Before mitosis, the two centrioles duplicate and two new daughter centrioles form adjacent to each preexisting maternal centriole. After initiation of daughter centriole synthesis, the procentrioles elongate in a process that is poorly understood. Here, we show that inhibition of cellular proteolysis by Z-L₃VS or MG132 induces abnormal elongation of daughter centrioles to approximately 4 times their normal length. This activity of Z-L₃VS or MG132 was found to correlate with inhibition of intracellular protease-mediated substrate cleavage. Using a small interfering RNA screen, we identified a total of nine gene products that either attenuated (seven) or promoted (two) abnormal Z-L₃VS–induced daughter centriole elongation. Our hits included known regulators of centriole length, including CPAP and CP110, but, interestingly, several proteins involved in microtubule stability and anchoring as well as centrosome cohesion. This suggests that nonproteasomal functions, specifically inhibition of cellular proteases, may play an important and underappreciated role in the regulation of centriole elongation. They also highlight the complexity of daughter centriole length control and provide a framework for future studies to dissect the molecular details of this process.     

From Zero to Many: Control of Centriole Number in Development and Disease

Centrioles are essential for the formation of microtubule-derived structures, including cilia, flagella and centrosomes. These structures are involved in a variety of functions, from cell motility to division. In most dividing animal cells, centriole formation is coupled to the chromosome cycle. However, this is not the case in certain specialized divisions, such as meiosis, and in some differentiating cells. For example, oocytes loose their centrioles upon differentiation, whereas multiciliated epithelial cells make several of those structures after they exit the cell cycle. Aberrations of centriole number are seen in many cancer cells. Recent studies began to shed light on the molecular control of centriole number, its variations in development, and how centriole number changes in human disease. Here we review the recent developments in this field.     

CP110 exhibits novel regulatory activities during centriole assembly in Drosophila

CP110 is a conserved centriole protein implicated in the regulation of cell division, centriole duplication, and centriole length and in the suppression of ciliogenesis. Surprisingly, we report that mutant flies lacking CP110 (CP110Δ) were viable and fertile and had no obvious defects in cell division, centriole duplication, or cilia formation. We show that CP110 has at least three functions in flies. First, it subtly influences centriole length by counteracting the centriole-elongating activity of several centriole duplication proteins. Specifically, we report that centrioles are ∼10% longer than normal in CP110Δ mutants and ∼20% shorter when CP110 is overexpressed. Second, CP110 ensures that the centriolar microtubules do not extend beyond the distal end of the centriole, as some centriolar microtubules can be more than 50 times longer than the centriole in the absence of CP110. Finally, and unexpectedly, CP110 suppresses centriole overduplication induced by the overexpression of centriole duplication proteins. These studies identify novel and surprising functions for CP110 in vivo in flies.     

PLK4 phosphorylation of CP110 is required for efficient centriole assembly

Centrioles are assembled during S phase and segregated into 2 daughter cells at the end of mitosis. The initiation of centriole assembly is regulated by polo-like kinase 4 (PLK4), the major serine/threonine kinase in centrioles. Despite its importance in centriole duplication, only a few substrates have been identified, and the detailed mechanism of PLK4 has not been fully elucidated. CP110 is a coiled-coil protein that plays roles in centriolar length control and ciliogenesis in mammals. Here, we revealed that PLK4 specifically phosphorylates CP110 at the S98 position. The phospho-resistant CP110 mutant inhibited centriole assembly, whereas the phospho-mimetic CP110 mutant induced centriole assembly, even in PLK4-limited conditions. This finding implies that PLK4 phosphorylation of CP110 is an essential step for centriole assembly. The phospho-mimetic form of CP110 augmented the centrosomal SAS6 level. Based on these results, we propose that the phosphorylated CP110 may be involved in the stabilization of cartwheel SAS6 during centriole assembly.     

CP110 cooperates with two calcium-binding proteins to regulate cytokinesis and genome stability

The centrosome is an integral component of the eukaryotic cell cycle machinery, yet very few centrosomal proteins have been fully characterized to date. We have undertaken a series of biochemical and RNA interference (RNAi) studies to elucidate a role for CP110 in the centrosome cycle. Using a combination of yeast two-hybrid screens and biochemical analyses, we report that CP110 interacts with two different Ca2+-binding proteins, calmodulin (CaM) and centrin, in vivo. In vitro binding experiments reveal a direct, robust interaction between CP110 and CaM and the existence of multiple high-affinity CaM-binding domains in CP110. Native CP110 exists in large (approximately 300 kDa to 3 MDa) complexes that contain both centrin and CaM. We investigated a role for CP110 in CaM-mediated events using RNAi and show that its depletion leads to a failure at a late stage of cytokinesis and the formation of binucleate cells, mirroring the defects resulting from ablation of either CaM or centrin function. Importantly, expression of a CP110 mutant unable to bind CaM also promotes cytokinesis failure and binucleate cell formation. Taken together, our data demonstrate a functional role for CaM binding to CP110 and suggest that CP110 cooperates with CaM and centrin to regulate progression through cytokinesis.     

Centriolar kinesin Kif24 interacts with CP110 to remodel microtubules and regulate ciliogenesis

We have identified a protein, Kif24, that shares homology with the kinesin-13 subfamily of motor proteins and specifically interacts with CP110 and Cep97, centrosomal proteins that play a role in regulating centriolar length and ciliogenesis. Kif24 preferentially localizes to mother centrioles. Loss of Kif24 from cycling cells resulted in aberrant cilia assembly but did not promote growth of abnormally long centrioles, unlike CP110 and Cep97 depletion. We found that loss of Kif24 leads to the disappearance of CP110 from mother centrioles, specifically in cycling cells able to form cilia. Kif24 is able to bind and depolymerize microtubules in vitro. Remarkably, ectopically expressed Kif24 specifically remodels centriolar microtubules without significantly altering cytoplasmic microtubules. Thus, our studies have identified a centriolar kinesin that specifically remodels a subset of microtubules, thereby regulating cilia assembly. These studies also suggest mechanistic differences between the regulation of microtubule elongation associated with centrioles and cilia.     

Centriole maturation and transformation to basal body

Centrioles and basal bodies are fascinating and mysterious organelles. They interconvert and seem to be crucial for a wide range of crucial cellular processes. However, intense research over the last years suggested that centrioles/basal bodies are essential mainly for the generation of cilia. Although a neglected organelle over a long time, interest in the primary cilia was recently rekindled by the notion that they are affected in a number of human diseases. Cilia formation is an intricate process that starts with the transformation of centrioles to basal bodies and their docking to the apical plasma membrane. Disturbance of basal body formation thus might cause ciliopathies. This review focuses on the formation of basal bodies in mammalian cells with an emphasis on basal bodies sprouting a primary cilium.     

Active Focal Length Control of Terahertz Slitted Plane Lenses by Magnetoplasmons

Active plasmonic devices are mostly designed at visible frequencies. Here, we propose an active terahertz (THz) plasmonic lens tuned by an external magnetic field. Unlike other tunable devices where the tuning is achieved by changing the plasma frequency of materials, the proposed active lens is tuned by changing the cyclotron frequency through manipulating magnetoplasmons (MPs). We have theoretically investigated the dispersion relation of MPs of a semiconductor?Cinsulator?Csemiconductor structure in the Voigt configuration and systematically designed several lenses realized with a doped semiconductor slab perforated with sub-wavelength slits. It is shown through finite?Cdifference time?Cdomain simulations that THz wave propagating through the designed structure can be focused to a small size spot via the control of MPs. The tuning range of the focal length under the applied magnetic field (up to 1?T) is ??3??, about 50% of the original focal length. Various lenses, including one with two focal spots and a tunable lens for dipole source imaging, are realized for the proposed structure, demonstrating the flexibility of the design approach. The proposed tunable THz plasmonic lenses may find applications in THz science and technology such as THz imaging.     

Cytoskeletal Control of Cell Length Regulation

It was shown that mouse embryo fibroblasts and human foreskin diploid fibroblasts of AGO 1523 line cultivated on specially prepared substrates with narrow (15 ± 3 m) linear adhesive strips were elongated and oriented along the strips, but the mean lengths of the fibroblasts of each type on the strips differed from those on the standard culture substrates. In contrast to the normal fibroblasts, the length of mouse embryonic fibroblasts with inactivated gene-suppressor Rb responsible for negative control of cell proliferation (MEF Rb-/-), ras-transformed mouse embryonic fibroblasts (MEF Rb-/-ras), or normal rat epitheliocytes of IAR2 line significantly exceeded those of the same cells on the standard culture substrates. The results of experiments with the drugs specifically affecting the cytoskeleton (colcemid and cytochalasin D) suggest that the constant mean length of normal fibroblasts is controlled by a dynamic equilibrium between two forces: centripetal tension of contractile actin-myosin microfilaments and centrifugal force generated by growing microtubules. This cytoskeletal mechanism is disturbed in MEF Rb-/- or MEF Rb-/-ras, probably, because of an impaired actin cytoskeleton and also in IAR2 epitheliocytes due to the different organization of the actin-myosin system in these cells, as compared to that in the fibroblasts.